Normal development in all multi-cellular life forms requires that specific genes be expressed in precise temporal and spatial patterns. Disrupting these patterns can cause mutant phenotypes and many disease states including cancer. We study the functions of modular DNA sequence elements (enhancers) that control when and where genes are expressed. Our work is focused on two enhancers from the Drosophila patterning gene even-skipped (eve). Each enhancer responds in a unique way to visible gradients of activator and repressor proteins that bind directly to sites within the enhancers. Despite intense study, specific questions remain about how these enhancers function. For example, it is not clear how enhancers sense specific levels of repressor proteins to establish sharp on/off borders of gene expression. It is also unclear how many different proteins are required for efficient activation, or how activator proteins interact after binding to the enhancer. Here we propose a series of experiments to address these important issues, with the following specific aims: First, we will study the role of putative DNA-binding sites in enhancer-mediated repression. This aim will focus on a global repression mechanism shared by at least two enhancers, and test the relationship between binding site affinity and enhancer sensitivity to repressor concentration in a well-defined system. Second, we will identify unknown cis-regulatory sequences that are critical for enhancer function using evolutionary sequence comparisons and computer-assisted search methods. Candidate sites will be tested by mutagenesis in reporter genes. Third, we will identify novel genes involved in the regulation of these enhancers using a combination of genetic experiments, bio-informatics approaches, and yeast one-hybrid screens. Finally, we will reconstitute specific aspects of enhancer activity using defined binding sites. The initial goal will be to define combinations of binding sites that can mediate enhancer-like activation at the correct time in embryogenesis. The completion of these studies will contribute significantly to our understanding of enhancer-mediated activation and repression, and provide insights into the mechanisms that cause some disease states. [unreadable] [unreadable]